The enzymatic isolation of cells and cell clusters from liver, pancreas, skin, cartilage, bone, neural tissue, and other organs has been accomplished and proven to be useful for various purposes including cellular characterization and implantation, e.g., isolation of islets of Langerhans (islets) from the pancreas for implantation in diabetic patients. Since collagen is a prominent structural protein within tissue, the enzyme collagenase is frequently used as a means of accomplishing the desired isolation.
Several forms of crude collagenase, e.g., crude bacterial collagenase derived from Clostridium histolyticum, are commercially available and have proven to be useful in isolating cells and cell clusters from tissue. These crude collagenases are actually a mixture of protease enzymes exhibiting collagenolytic and proteolytic activity and non-protease components, including fermentation by-products, fermentation media, pigment, and other enzymes (e.g. phospholipase). These non-protease components of crude collagenase are generally recognized by those skilled in the art as being inert, that is, not affecting the activity of the protease enzymes. Unfortunately, these commercially available crude collagenases have been shown to contain varying amounts of the protease and non-protease components, thereby causing substantial variations in efficacy between lots. In addition to variability problems, use of these crude collagenases in research has resulted in poor cell integrity, low cell number, and fragmented cell clusters. These problems are significant, especially when the isolated cells or cell clusters are subsequently transplanted. For example, it has been shown that the efficacy of the transplanted islet mass, i.e. its ability to produce insulin in a host, is greatly diminished with reduced size and number. The importance of the protease enzymes to the process of obtaining an efficacious cellular isolation, i.e. maintaining cellular integrity, isolating larger cell clusters and isolating more cells or cell clusters, has been recognized by those skilled in the art. Specifically, the presence of collagenase Class I (collagenase I) and collagenase Class II (collagenase II) enzymes and the presence of a neutral protease have been found to influence the efficacy of the cellular isolation. The protease clostripain has been reported to have a negative influence on certain cell isolations (Hefley, J Bone and Mineral Res. 2:6, 1987, pp.505-516). However, while many of the problems associated with obtaining a pure cell isolation have been defined, appropriate remedies have heretofore remained undiscovered.